US9506090B2 - Method for synthesizing FDCA and derivates thereof - Google Patents

Method for synthesizing FDCA and derivates thereof Download PDF

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US9506090B2
US9506090B2 US14/033,300 US201314033300A US9506090B2 US 9506090 B2 US9506090 B2 US 9506090B2 US 201314033300 A US201314033300 A US 201314033300A US 9506090 B2 US9506090 B2 US 9506090B2
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ddg
fdca
acid
derivative
conversion
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US20140106414A1 (en
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Spiros Kambourakis
Benjamin M. Griffin
Kevin V. Martin
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Viridos Inc
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Synthetic Genomics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/58—Aldonic, ketoaldonic or saccharic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • furan-2,5-dicarboxylic acid also known as 2,5-furandicarboxylic acid, hereinafter abbreviated as FDCA
  • FDCA furan-2,5-dicarboxylic acid
  • TPA terephthalic acid
  • the present invention provides methods for producing a product of one or more enzymatic pathways.
  • the pathways used in the methods of the invention involve one or more conversion steps such as, for example, an enzymatic conversion of guluronic acid into D-glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid Step 7b); and an enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16).
  • the methods of the invention produce 2,5-furandicarboxylic acid (FDCA) as a product.
  • FDCA 2,5-furandicarboxylic acid
  • the methods include both enzymatic and chemical conversions as steps.
  • Various pathways are also provided for converting glucose into 5-dehdyro-4-deoxy-glucarate (DDG), and for converting glucose into FDCA.
  • DDG 5-dehdyro-4-deoxy-glucarate
  • the methods can also involve the use of engineered enzymes that perform reactions with high specificity and efficiency.
  • the invention provides a method for producing a product of an enzymatic or chemical pathway from a starting substrate.
  • the pathway can contain any one or more of the following conversion steps: an enzymatic conversion of guluronic acid into D-glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid (Step 7b); and an enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); an enzymatic conversion of 1,5-gluconolactone to gulurono-lactone (Step 19).
  • the product of the enzymatic pathway is 5-dehydro-4-deoxy-glucarate (DDG).
  • the substrate of the method can be glucose, and the product can 5-dehydro-4-deoxy-glucarate (DDG).
  • the method can involve the steps of the enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); the enzymatic conversion of 1,5-gluconolactone to gulurono-lactone (Step 19); the enzymatic conversion of gulurono-lactone to guluronic acid (Step 1B); the enzymatic conversion of guluronic acid to D-glucarate (Step 7); and the enzymatic conversion of D-glucarate to 5-dehydro-4-deoxy-glucarate (DDG) (Step 8).
  • the substrate is glucose and the product is DDG
  • the method involves the steps of the conversion of D-glucose to 1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone to gluconic acid (Step 1a); the conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of 5-ketogluconate (5-KGA) to L-Iduronic acid (Step 15); the conversion of L-Iduronic acid to Idaric acid (Step 7b); and the conversion of Idaric acid to DDG (Step 8a).
  • the substrate is glucose and the product is DDG and the method involves the steps of the conversion of D-glucose to 1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone to gluconic acid (Step 1a); the conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of 5-ketogluconate (5-KGA) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); the conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 4); and the conversion of 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG (Step 5).
  • the substrate is glucose and the product is DDG
  • the method involves the steps of: the conversion of D-glucose to 1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone to gluconic acid (Step 1a); the conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of 5-ketogluconate (5-KGA) to L-Iduronic acid (Step 15); the conversion of L-Iduronic acid to 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 7B); and the conversion of 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG (Step 5).
  • Any of the methods disclosed herein can further involve the step of converting the DDG to 2,5-furan-dicarboxylic acid (FDCA). Converting the DDG to FDCA in any of the methods can involve contacting DDG with an inorganic acid to convert the DDG to FDCA.
  • FDCA 2,5-furan-dicarboxylic acid
  • the invention provides a method for synthesizing derivatized (esterified) FDCA.
  • the method involves contacting DDG with an alcohol, an inorganic acid at a temperature in excess of 60 C to form derivatized FDCA.
  • the alcohol is methanol, butanol or ethanol.
  • the invention provides a method for synthesizing a derivative of FDCA.
  • the method involves contacting DDG with an alcohol, an inorganic acid, and a co-solvent to produce a derivative of DDG; optionally purifying the derivative of DDG; and contacting the derivative of DDG with an inorganic acid to produce a derivative of FDCA.
  • the inorganic acid can be sulfuric acid and the alcohol can be ethanol or butanol.
  • the co-solvent can be any of THF, acetone, acetonitrile, an ether, butyl acetate, an dioxane, chloroform, methylene chloride, 1,2-dichloroethane, a hexane, toluene, and a xylene.
  • the derivative of DDG is di-ethyl DDG and the derivative of FDCA is di-ethyl FDCA
  • the derivative of DDG is di-butyl DDG and the derivative of FDCA is di-butyl FDCA.
  • the invention provides a method for synthesizing FDCA.
  • the method involves contacting DDG with an inorganic acid in a gas phase.
  • the invention provides a method for synthesizing FDCA.
  • the method involves contacting DDG with an inorganic acid at a temperature in excess of 120 C.
  • the invention provides a method for synthesizing FDCA.
  • the method involves contacting DDG with an inorganic acid under anhydrous reaction conditions.
  • FIG. 1 is a electrophoretic gel of crude lysates and purified enzymes of proteins 474, 475, and 476.
  • FIGS. 2 a - h is a schematic illustration of the pathways of Routes 1, 2, 2A, 2C, 2D, 2E, 2F, respectively.
  • FIGS. 3 a - c present a schematic illustration of the pathways of Routes 3, 4, and 5, respectively.
  • FIG. 4 is an HPCL-MS analysis of the dehydration of gluconate with gluconate dehydratase to produce DHG by pSGI-359.
  • FIG. 5 is a graphical illustration of semicarbizide assay plots for measuring the activity of gluconate dehydratases.
  • FIGS. 6 a and 6 b provide Lineweaver-Burk plots for the oxidation of glucuronate and iduronate with three enzymes of the invention.
  • FIG. 7 a shows the results of an HPLC analysis of time points for the isomerization of 5KGA and Iduronate using enzymes DTHU isomerases in the EC 5.3.1.17 family.
  • FIG. 7 b shows an HPLC analysis of time points for the isomerization of 5KGA and iduronate using enzymes in the EC 5.3.1.17 family.
  • FIG. 8 shows product formation for the isomerization of 5KGA and iduronate with enzymes in the EC 5.3.1.n1 family. The data were obtained from enzymatic assays.
  • FIG. 9 HPLC analysis of the formation of 2,5-DDH and the reduction of 5 KGA concentration over time. Total ion counts for 2,5-DDH are shown.
  • FIG. 10 is a HPLC-MS chromatogram showing the production of guluronic acid lactone from 1,5-gluconolactone. An overlay of a trace of authentic guluronic acid is shown.
  • FIG. 11 is a schematic illustration of the Scheme 6 reaction pathway.
  • FIGS. 12 a and 12 b are LC-MS chromatograms showing S-KGA and DDG reaction products, respectively.
  • FIG. 13 is a an LC-MS chromatogram showing FDCA and FDCA dibutyl ester derivative reaction products.
  • FIG. 14 a is a GC-MS analysis of a crude reaction sample of the diethyl-FDCA synthesis from the reaction of DDG with ethanol. Single peak corresponded to diethyl-FDCA.
  • FIG. 14 b is an MS fragmentation of the major product from the reaction of DDG with ethanol.
  • FIG. 15 a is a GC-MS analysis of a crude reaction sample of the diethyl-FDCA synthesis from the reaction of DDG with ethanol. Single peak corresponded to diethyl-FDCA.
  • FIG. 15 b is a MS fragmentation of the major product from the reaction of DDG with ethanol.
  • FIG. 16 is a schematic illustration of the synthesis of FDCA and its derivatives from DTHU.
  • FIG. 17 is a schematic illustration of Scheme 1.
  • Enzymes are ST-1: glucose oxidase; ST-1A: hydrolysis-chemical; ST-14: gluconate dehydrogenase (pSGI-504); ST-15: 5-dehydro-4-deoxy-D-glucuronate isomerase (DTHU IS, pSGI-434); ST-7B: Uronate dehydrogenase (UroDH, pSGI-476)); ST-8A Glucarate dehydratase (GlucDH, pSGI-353); ST-A: NAD(P)H oxidase (NADH_OX, pSGI-431); ST-B: Catalase.
  • FIG. 17 b shows the concentration of reaction intermediates over the first 3 h as analyzed by HPLC. Formation of DDG is shown in both reactions.
  • the present invention provides methods for producing a product of an enzymatic pathway.
  • the methods can comprise the enzymatic conversion of a substrate into a product.
  • FDCA 2,5-furanyl dicarboxylic acid
  • the methods can comprise one or more enzymatic and/or chemical substrate-to-product conversion steps disclosed herein.
  • the pathways of the invention are comprised of one or more steps. It is understood that a step of a pathway of the invention can involve the forward reaction or the reverse reaction, i.e., the substrate A being converted into intermediate B and product C, while in the reverse reaction substrate C is converted into intermediate B and product A. In the methods both the forward and the reverse reactions are described as the step unless otherwise noted.
  • the methods involve producing a product of a pathway, which can be an enzymatic pathway.
  • the pathways can include one or more chemical steps.
  • the methods involve one or more enzymatic and/or chemical conversion steps, which convert a substrate to a product.
  • Steps that can be included in the methods include, for example, any one or more of: an enzymatic conversion of guluronic acid into D-glucarate (Step 7); an enzymatic conversion of L-iduronic acid to Idaric acid (7B); an enzymatic conversion of L-Iduronic acid to 4-deoxy-5-threo-hexosulose uronate (DTHU)(7B); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid Step 7B); and an enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo hexano
  • An enzymatic step or pathway is a step or pathway that requires an enzyme as a catalyst in the reaction to make the step proceed.
  • Chemical steps can be performed without an enzyme as a catalyst in the reaction.
  • Any one or more of the steps recited in the methods can be an enzymatic step. In some embodiments every step of the pathway is an enzymatic step, while in other embodiments one or more steps in the pathway is a chemical step.
  • any of the methods can include a step involving the addition of the substrate of the reaction to a reaction mix containing the enzyme that performs the conversion.
  • the method of converting guluronic acid into D-glucarate can involve the addition of guluronic acid as starting substrate to the reaction mix;
  • the enzymatic conversion of L-iduronic acid to Idaric acid (7B) can involve the addition of L-Iduronic acid as starting substrate to the reaction mix;
  • the enzymatic conversion of L-Iduronic acid to 4-deoxy-5-threo-hexosulose uronate (DTHU) (7B) can involve the addition of DTHU as starting substrate to the reaction mix.
  • Another step that can be included in any of the methods is a step of purifying from the reaction mixture a reaction product.
  • a step of purifying D-glucarate or L-Iduronic acid, or Idaric acid, or 4,6-dihydroxy 2,5-diketo hexanoate can be included in any of the methods described herein.
  • Any of the methods disclose can include a step of isolating or purifying DDG or FDCA from the reaction mixture.
  • the reaction mix used in the methods can be a cell lysate of cells that contain one or more enzymes that perform the enzymatic conversion, but can also be a reaction mixture containing components added by the user to form a reaction mixture, or can contain components purified from a cell lysate, or may be contained in a whole cell biocatalyst.
  • the methods of the invention are methods of converting glucose to DDG, or glucose to FDCA, or glucose to DTHU or DEHU, or for converting DDG to FDCA.
  • the methods can involve converting the starting substrate in the method into the product.
  • the starting substrate is the chemical entity considered to begin the method and the product is the chemical entity considered to be the final end product of the method.
  • Intermediates are those chemical entities that are created in the method (whether transiently or permanently) and that are present between the starting substrate and the product.
  • the methods and pathways of the invention have about four or about five intermediates or 4-5 intermediates, or about 3 intermediates, or 3-5 intermediates, or less than 6 or less than 7 or less than 8 or less than 9 or less than 10 or less than 15 or less than 20 intermediates, meaning these values not counting the starting substrate or the final end product.
  • the invention provides methods of producing FDCA and/or DDG, from glucose that have high yields.
  • the theoretical yield is the amount of product that would be formed if the reaction went to completion under ideal conditions.
  • the methods of the invention produce DDG from glucose, fructose, or galactose with a theoretical yield of at least 50% molar, or at least 60% molar or at least 70% molar, or at least 80% molar, at least 90% molar or at least 95% molar or at least 97% molar or at least 98% molar or at least 99% molar, or a theoretical yield of 100% molar.
  • the methods of the invention also can provide product with a carbon conservation of at least 80% or at least 90% or at least 99% or 100%, meaning that the particular carbon atoms present in the initial substrate are present in the end product of the method at the recited percentage.
  • the methods produce DDG and/or FDCA from glucose via dehydration reactions.
  • the invention also provides specific pathways for synthesizing and producing a desired product. Any of the following described routes or pathways can begin with glucose and flow towards a desired product.
  • D-glucose is the starting substrate and the direction of the pathway towards any intermediate or final product of the pathway is considered to be in the downstream direction, while the opposite direction towards glucose is considered the upstream direction.
  • routes or pathways can flow in either the downstream or upstream direction.
  • glucose, fructose, galactose, or any intermediate in any of the pathways can be the starting substrate in a method of the invention, and DDG, FDCA, or any intermediate in any of the routes or pathways of the invention can be the final end product of a method of the invention.
  • the disclosed methods therefore include any one or more steps disclosed in any of the routes or pathways of the invention for converting any starting substrate or intermediate into any end product or intermediate in the disclosed routes or pathways using one or more of the steps in the disclosed routes or pathways.
  • the methods can be methods for converting glucose to DDG, or glucose to guluronic acid, or glucose to galactarate, or glucose to DTHU, or glucose to DEHU, or for converting glucose to guluronic acid, or for converting glucose to iduronic acid, or for converting glucose to idaric acid, or for converting glucose to glucaric acid, or for converting galactarate to DDG, or for converting guluronic acid to D-glucarate, or for converting 5-KGA to L-Iduronic acid, or for converting L-Iduronic acid to Idaric acid, or for converting 5-KGA to 2,5-DDH or DTHU, or for converting DHG to DEHU.
  • the methods utilize the steps disclosed in the methods and pathways of the invention
  • Route 1 is illustrated in FIG. 2 a .
  • Route 1 converts D-glucose (or any intermediate in the pathway) into 5-dehydro-4-deoxy-glucarate (DDG) via an enzymatic pathway via a series of indicated steps.
  • Route 1 converts D-glucose into DDG via a pathway having 1,5-gluconolactone, gluconic acid, 3-dehydro-gluconic acid (DHG), 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH), and 4-deoxy-L-threo-hexosulose uronate (DTHU) as intermediates and DDG as the final end product.
  • DTHU 4-deoxy-L-threo-hexosulose uronate
  • the steps are the enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); the enzymatic conversion of 1,5-gluconolactone to gluconic acid (Step 1A); the enzymatic conversion of gluconic acid to 3-dehydro-gluconic acid (DHG) (Step 2); the enzymatic conversion of 3-dehydro-gluconic acid (DHG) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 3); the enzymatic conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-L-threo-hexosulose uronate (DTHU) (Step 4); and the enzymatic conversion of 4-deoxy-L-threo-hexosulose uronate (DTHU) to 5-dehydro-4-deoxy glucarate (DDG) (Step 5).
  • Route 2 is illustrated in FIG. 2 b and converts D-glucose into DDG.
  • the steps in the Route 2 pathway are the enzymatic conversion of D-glucose into 1,5-gluconolactone (Step 1); the enzymatic conversion of 1,5-gluconolactone to gluconic acid (Step 1A); the enzymatic conversion of gluconic acid to guluronic acid (Step 6); the enzymatic conversion of guluronic acid to D-glucarate (Step 7); the enzymatic conversion of D-glucarate to DDG (Step 8).
  • Route 2 also comprises sub-routes where glucose or any intermediate in the pathway is converted into any other downstream intermediate as final product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 2A is illustrated in FIG. 2 c .
  • the steps in Route 2A are the enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); the enzymatic conversion of 1,5-gluconolactone to guluronic acid lactone (Step 19); the enzymatic conversion of guluronic acid lactone to guluronic acid (Step 1B); the enzymatic conversion of guluronic acid to D-glucarate (Step 7); the enzymatic conversion of D-glucarate to 5-dehydro-4-deoxy-glucarate (DDG) (Step 8).
  • Route 2A also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 2B is illustrated in FIG. 2 d .
  • the steps in Route 2B are the enzymatic conversion of D-glucose into gluconic acid (Steps 1 and 1A); the enzymatic conversion of gluconic acid into 5-ketogluconate (5-KGA) (Step 14); the enzymatic conversion of 5-KGA into L-Iduronic acid (Step 15); the enzymatic conversion of L-Iduronic acid into Idaric acid (Step 7B); the enzymatic conversion of Idaric acid into DDG (Step 8A).
  • Route 2B also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 2C is illustrated in FIG. 2 e .
  • the steps in Route 2C are the enzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the enzymatic conversion of 5-KGA to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); the enzymatic conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 4); the enzymatic conversion of DTHU to DDG (Step 5).
  • Route 2C also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 2D is illustrated in FIG. 2 f .
  • the steps in Route 2D are the enzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the enzymatic conversion of 5-KGA to Iduronic acid (Step 15); the enzymatic conversion of L-Iduronic acid to DTHU (Step 17); the enzymatic conversion of DTHU to DDG (Step 5).
  • Route 2D also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 2E is illustrated in FIG. 2 g .
  • the steps in Route 2D are the enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); the enzymatic conversion of 1,5-gluconolactone to guluronic acid lactone (Step 19); the enzymatic conversion of guluronic acid lactone to guluronic acid (Step 1B); the enzymatic conversion of guluronic acid to 4-deoxy-erythro-hexosulose uronate (DEHU) (Step 17A); the enzymatic conversion of DEHU to 3-deoxy-D-erythro-2-hexylosaric acid (DDH) (Step 7A).
  • Route 2E also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 2F is illustrated in FIG. 2 h .
  • the steps in Route 2F are the enzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic conversion of gluconic acid to guluronic acid (Step 6); the enzymatic conversion of guluronic acid to 4-deoxy-erythro-hexosulose uronate (DEHU) (Step 17); the enzymatic conversion of DEHU to 3-deoxy-D-erythro-2-hexulosaric acid (DDH) (Step 7A).
  • Route 2F also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 3 is illustrated in FIG. 3 a .
  • the steps in Route 3 are the enzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic conversion of gluconic acid to 3-dehydro-gluconic acid (DHG) (Step 2); the enzymatic conversion of DHG to 4-deoxy-erythro-hexosulose uronate (DEHU) (Step 6A); the enzymatic conversion of DEHU to DDG (Step 7A).
  • Route 3 also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 4 is illustrated in FIG. 3 b .
  • the steps in Route 4 are the enzymatic conversion of D-glucose to ⁇ -D-gluco-hexodialdo-1,5-pyranose (Step 9); the enzymatic conversion of ⁇ -D-gluco-hexodialdo-1,5-pyranose to ⁇ -D-glucopyranuronic acid (Step 10); the enzymatic conversion of ⁇ -D-glucopyranuronic acid to D-glucaric acid 1,5-lactone (Step 11); the enzymatic conversion of D-glucaric acid 1,5-lactone to D-glucarate (Step 1C); the enzymatic conversion of D-glucarate to DDG (Step 8).
  • Route 4 also comprises sub-routes where glucose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final end product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • Route 5 is illustrated in FIG. 3 c .
  • the steps in Route 5 are the enzymatic conversion of D-galactose to D-galacto-hexodialdose (Step 9A); the enzymatic conversion of D-galacto-hexodialdose to galacturonate (Step 10A); the enzymatic conversion of galacturonate to galactarate (Step 11A); the enzymatic conversion of galactarate to DDG (Step 13).
  • Route 5 also comprises sub-routes where galactose or any intermediate in the pathway as starting substrate is converted into any other downstream intermediate as final product, and each sub-route is considered disclosed as if each is set forth herein in full.
  • enzymes and nucleic acids that encode the enzymes
  • additional enzymes or nucleic acids encoding the enzymes having a sequence identity to any enzyme or member of a class of enzymes disclosed herein will also be useful in the invention that has a sequence identity of at least 40% or at least 50% or at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% to any enzyme or member of an enzyme class disclosed herein.
  • Percent sequence identity or homology with respect to amino acid or nucleotide sequences is defined herein as the percentage of amino acid or nucleotide residues in the candidate sequence that are identical with the known polypeptides, after aligning the sequences for maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent identity or homology.
  • Homology or identity at the nucleotide or amino acid sequence level may be determined using methods known in the art, including but not limited to BLAST (Basic Local Alignment Search Tool) analysis using the algorithms employed by the programs blastp, blastn, blastx, tblastn and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci.
  • a functional fragment of any of the enzymes (or nucleic acids encoding such enzymes) disclosed herein may also be used.
  • the term “functional fragment” refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, where the remaining amino acid sequence has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the corresponding positions in the reference sequence, and that retains about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the full-length polypeptide.
  • Functional fragments may comprise, e.g., 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, or 20% or less of the full-length polypeptide, and can include, for example, up to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length polypeptide.
  • the EC numbers provided use the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology.
  • Step 1 Conversion (oxidation or dehydrogenation) of glucose to 1,5-gluconolactone.
  • This step can be performed with various enzymes, such as those of the family oxygen dependent glucose oxidases (EC 1.1.3.4) or NAD(P)-dependent glucose dehydrogenases (EC 1.1.1.118, EC 1.1.1.119).
  • Gluconobacter oxydans has been shown to efficiently oxidize glucose to gluconic acid and 5-ketogluconate (5-KGA) when grown in a fermentor.
  • Enzymes of the family of soluble and membrane-bound PQQ-dependent enzymes EC 1.1.99.35 and EC 1.1.5.2 found in Gluconobacter and other oxidative bacteria can be used.
  • Quinoprotein glucose is another enzyme that is useful in performing this step. The specific enzyme selected will be dependent on the desired reaction conditions and necessary co-factors that will be present in the reaction, which are illustrated in Table 1.
  • Step 1A Conversion (e.g., hydrolysis) of 1,5-gluconolactone to gluconate.
  • This step can be performed chemically in aqueous media and the rate of hydrolysis is dependent on pH (Shimahara, K, Takahashi, T., Biochim. Biophys. Acta (1970), 201, 410). Hydrolysis is faster in basic pH (e.g. pH 7.5) and slower in acid pH.
  • Many microorgranisms also contain specific 1,5-glucono lactone hydrolases, and a few of them have been cloned and characterized (EC 3.1.1.17; Shinagawa, E Biosci. Biotechnol. Biochem. 2009, 73, 241-244).
  • Step 1B Conversion of Guluronic acid lactone to guluronic acid.
  • the chemical hydrolysis of guluronic acid lactone can be done by a spontaneous reaction in aqueous solutions.
  • An enzyme capable of catalyzing this hydrolysis is identified amongst the large number of lactonases (EC 3.1.1.XX and more specifically 3.1.1.17, 3.1.1.25).
  • Step 2 Conversion of gluconic acid to 3-dehydro gluconic acid (DHG):
  • gluconate dehydratases can be used in the dehydration of gluconic acid to dehydro gluconic acid (DHG). Examples include those belonging to the gluconate dehydratase family (EC 4.2.139). A specific example of such a dehydratase has been shown to dehydrate gluconate (Kim, S. Lee, S. B. Biotechnol. Bioprocess Eng . (2008), 13, 436). Particular examples of enzymes from this family and their cloning are shown in Example 1.
  • Step 3 Conversion of 3-dehydro-gluconic acid (DHG) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH). Enzymes, 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (or DHG dehydrogenases) (EC 1.1.1.127) for performing this conversion have been described.
  • Step 4 Conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-L-threo-hexosulose uronate (DTHU).
  • Enzymes of the family EC 5.3.1.12 can be used in this step, and Step 15 shows that five such enzymes were cloned and shown to have activity for the dehydration of 5-KGA. These enzyme will also show activity towards 2,5-DDH and DTHU.
  • Step 5 Conversion of DTHU to 5-dehydro-4-deoxy-glucarate (DDG).
  • DDG can be produced from the chemical or enzymatic oxidation of DTHU, for example with a mild chemical catalyst capable of oxidizing aldehydes in the presence of alcohols. Aldehyde oxidases can be used to catalyze this oxidation. Oxidative bacteria such as Acetobacter and Gluconobacter (Hollmann et al Green Chem. 2011, 13, 226) will be useful in screening.
  • Enzymes of the following families can perform this reaction: aldehyde oxidase EC1.2.3.1, aldehyde ferredoxin oxidoreductase (EC1.2.7.5), and in all the families of EC1.2.1.-XX. Enzymes of the family of uronate dehydrogenases (EC 1.1.1.203) (e.g. see Step 7) will also have this activity. Other enzymes with both alcohol and aldehyde oxidation activity can be used, including enzymes in the alditol oxidase family (see Steps 19 and 6). Other broad substrate oxidases include soluble and membrane bound PQQ-dependent alcohol/aldehyde oxidases.
  • periplasmic PQQ oxidases enzymes and their homologs belonging into Type I (EC 1.1.91) and II (EC 1.1.2.8) families as well as membrane bound PQQ oxidases belonging into EC 1.1.5.X families are useful.
  • aldehyde dehydrogenases/oxidases that act on DTHU can be used.
  • Steps 6 and 6A Conversion of gluconic acid to guluronic acid (6) and conversion of 3-dehydro-gluconic acid (DHG) to 4-deoxy-5-erythro-hexosulose uronate (DEHU)(6A).
  • the enzymes described in Step 5 are useful for these conversions.
  • Other useful enzymes include NAD(P)-dependent dehydrogenases in the EC 1.1.1.XX families and more specifically glucuronate dehydrogenase (EC 1.1.1.19), glucuronolactone reductase (EC 1.1.1.20).
  • O 2 -dependent alcohol oxidases with broad substrate range including sugars will be useful (EC 1.1.3.XX), including sorbitol/mannitol oxidases (EC 1.1.3.40), hexose oxidases (EC 1.1.3.5), alcohol oxidases (EC 1.1.3.13) and vanillin oxidase (EC 1.1.3.38).
  • PQQ-dependent enzymes and enzymes present in oxidative bacteria can also be used for these conversions.
  • Steps 7 and 7B Conversion of guluronic acid to D-glucaric acid (7) and conversion of L-Iduronic acid to idaric acid (7B). These steps can be accomplished with enzymes of the family of uronate dehydrogenases (EC 1.1.1.203) or the oxidases, as described herein.
  • Step 7A Conversion of 4-deoxy-5-erythro-hexosulose uronate (DEHU) to 3-deoxy-D-erythro-2-hexylosaric acid (DDH). The Same enzymes described in Step 5 will be useful for performing this conversion.
  • Steps 8 and 8A Conversion of D-glucaric acid to 5-dehydro-4-deoxy-glucarate (DDG) (Step 8) and conversion of Idaric acid to DDG (Step 8A).
  • Enzymes of the family of glucarate dehydratases (EC 4.2.1.40) can be used to perform these steps. Enzymes of this family have been cloned and have been shown to efficiently convert glucarate to DDG.
  • Two D-glucarate dehydratases (EC 4.2.1.40) were cloned as shown in the Table of cloned glucarate dehydratases below. Both enzymes showed very high activity for the dehydration of Glucarate to DDG using the semicarbazide assay, as described in Step 2.
  • Step 9 and 9A Conversion of ⁇ -glucose to ⁇ -D-gluco-hexodialdo-1,5-pyranose (9) and conversion of D-galactose to D-galacto-hexodialdose (9A).
  • Oxidases such as those of the galactose oxidase family (EC 1.1.3.9) can be used in this step. Mutant galactose oxidases are also engineered to have activity on glucose and have been described (Arnold, F. H. et al ChemBioChem, 2002, 3(2), 781).
  • Step 10 Conversion of ⁇ -D-gluco-hexodialdo-1,5-pyranose to ⁇ -D-glucopyranuronic acid (step 10) and D-galacto-hexodialdose to galacturonate (10A).
  • This step can be performed using an enzyme of the family of aldehyde dehydrogenases.
  • Step 11 and 11A Conversion of ⁇ -D-glucopyranuronic acid to glucuronic acid 1,5-lactone.
  • Aldehyde dehydrogenases and oxidases as described in Step 5 will be useful in performing this step.
  • Uronate dehydrogenases described in Steps 7 and 7B can also be useful in performing this step.
  • Step-11A is the conversion of galacturonate to galactarate.
  • the uronate dehydrogenase (EC 1.1.1.203), for example those described in Steps 7 and 7B, will be useful in performing this step.
  • Step 12 Conversion of fructose to glucose. Glucose and fructose isomerases (EC 5.3.1.5) will be useful in performing this step.
  • Step 13 Conversion of galactarate to 5-dehydro-4-deoxy-D-glucarate (DDG). Enzymes of the family of galactarate dehydrogenases (EC 4.2.1.42) can be used to perform this step, and additional enzymes can be engineered for performing this step.
  • Step 14 Conversion of gluconate to 5-ketogluconate (5-KGA).
  • a number of enzymes of the family of NAD(P)-dependent dehydrogenases (EC1.1.1.69) have been cloned and shown to have activity for the oxidation of gluconate or the reduction of 5KGA.
  • the NADPH-dependent gluconate 5-dehydrogenase from Gluconobacter (Expasy P50199) was synthesized for optimal expression in E. coli as shown herein and was cloned in pET24 (pSGI-383). The enzyme was expressed and shown to have the required activities.
  • Additional enzymes useful for performing this step include those of the family of PQQ-dependent enzymes present in Gluconobacter (Peters, B. et al. Appl. Microbiol Biotechnol ., (2013), 97, 6397), as well as the enzymes described in Step 6. Enzymes from these families can also be used to synthesize 5KGA from gluconate.
  • Step 15 Conversion of 5-KGA to L-Iduronic acid. This step can be performed with various enzymes from different isomerase families, as further described in Example 4.
  • Step 16 Conversion of 5-KGA to (4S)-4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH). This dehydration can be performed with enzymes in the gluconate dehydratase family (EC 4.2.3.39), such as those described in Example 5 or Step 17.
  • Step 17 and 17A L-Iduronate to 4-deoxy-5-threo-hexosulose uronate (DTHU) and Guluronate to 4-deoxy-5-hexoulose uronate (DHU).
  • Enzymes of the family of dehydratases are identified that can be used in the performance of this step. Enzymes from the families of gluconate or glucarate dehydratases will have the desired activity for performing these steps. Furthermore, many dehydratases of the family (EC 4.2.1.X) will be useful in the performance of these steps.
  • enzymes that dehydrate 1,2-dyhydroxy acids to selectively produce 2-keto-acids will be useful, such as enzymes of the families: EC 4.2.1.6 (galactonate dehydratase), EC 4.2.1.8 (mannonate dehydratase), EC 4.2.1.25 (arabonate dehydratase), EC 4.2.1.39 (gluconate dehydratase), EC 4.2.1.40 (glucarate dehydratase), EC 4.2.1.67 (fuconate dehydratase), EC 4.2.1.82 (xylonate dehydratase), EC 4.2.1.90 (rhamnonate dehydratase) and dihydroxy acid dehydratases (4.2.1.9). Since known enzyme selectivity is the production of an alpha-keto acid the identified enzymes will produce DEHU and DTHU, respectively, as the reaction products.
  • Step 19 Conversion of 1,5-gluconolactone to guluronic acid lactone. This step can be performed by enzymes of the family of alditol oxidases (EC 1.1.3.41) or the enzymes described in Step 6.
  • the present invention also provides novel methods of converting DDG to FDCA and FDCA esters.
  • Esters of FDCA include diethyl esters, dibutyl esters, and other esters.
  • the methods involve converting DDG into a DDG ester by contacting DDG with an alcohol, an inorganic acid, and optionally a co-solvent to produce a derivative of DDG.
  • the alcohol can be methanol, ethanol, propanol, butanol, or any C1-C20 alcohol.
  • the inorganic acid can be sulfuric acid.
  • the co-solvent can be any of or any mixture of THF, acetone, acetonitrile, an ether, butyl acetate, an dioxane, chloroform, methylene chloride, 1,2-dichloroethane, a hexane, toluene, and a xylene.
  • the esterified DDG can then be converted into esterified FDCA.
  • the DDG can be optionally purified as a step prior to performing the method.
  • Purifying the DDG can comprise removing water from the solvent comprising the DDG, for example removing greater than 87% of the water or greater than 90% of the water or greater than 95% of the water or greater than 97% or greater than 98% or greater than 99% of the water from the solvent comprising the DDG. Yields of greater than 25% or 30% or 35% or 40% or 45% molar can be obtained.
  • DDG purification for dehydration or esterification was performed by acidifying the DDG, e.g., by lowering the pH of the reaction with the addition of conc HCl to pH ⁇ 2.5. At this pH proteins and any residual glucarate precipitate are removed by filtration and the mixture is lyophilized to give a white powder consisting of DDG and the reaction salts.
  • This DDG can be dehydrated to give 2,5-FDCA, or be esterified to dibutyl-DDG (or di-ethyl DDG) prior to dehydration.
  • This method of purifying or esterifying DDG can be added as a step in any of the methods and pathways disclosed herein that produce DDG.
  • the invention also provides various methods of synthesizing FDCA.
  • One method for synthesizing FDCA involves contacting DDG with an alcohol, an inorganic acid at a high temperature to form FDCA.
  • the alcohol can be any alcohol, and examples include (but are not limited to) methanol, ethanol, propanol, and butanol. Diols can also be used.
  • the high temperature can be a temperature greater than 70° C. or greater than 80° C. or greater than 90° C. or greater than 100° C. or greater than 110° C. or greater than 120° C. or greater than 130° C. or greater than 140° C. or greater than 150° C. to form FDCA. Reaction yields of greater than 20% or greater than 30% or greater than 35% or greater than 40% can be achieved.
  • the invention also provides methods for synthesizing derivatives of FDCA.
  • the methods involve contacting a derivative of DDG with an inorganic acid to produce a derivative of FDCA.
  • the inorganic acid can be, for example, sulfuric acid.
  • the derivative of DDG can be purified prior to contacting it with the second inorganic acid.
  • Non-limiting examples of the derivative of DDG that can be used include methyl DDG, ethyl DDG, propyl DDG, butyl DDG, isobutyl DDG, di-methyl DDG, di-ethyl DDG, di-propyl DDG, di-butyl DDG.
  • the derivative of FDCA produced can be methyl FDCA, ethyl FDCA, propyl FDCA, butyl FDCA, di-methyl FDCA, di-ethyl FDCA, di-propyl FDCA, di-butyl FDCA, and isobutyl FDCA.
  • the derivate of FDCA produced corresponds to the derivative of DDG used in the method.
  • the derivative of FDCA can then be de-esterified to produce FDCA.
  • the method can also be conducted in the gas phase, e.g., using the parameters described below.
  • Another method for synthesizing FDCA or derivatives of FDCA involves contacting DDG or derivatives of DDG (any described herein) with an inorganic acid in a gas phase, which can be done with a short residence time, e.g., of less than 10 seconds or less than 8 seconds, or less than 6 seconds or less than 5 seconds or less than 4 seconds or less than 3 seconds or less than 2 seconds or less than 1 second.
  • the residence time refers to the time that the sample is present in the reaction zone of the high temperature flow through reactor.
  • the method can also be conducted at high temperatures, for example at temperatures greater than 150° C., greater than 200° C., greater than 250° C., greater than 300° C. or greater than 350° C.
  • Another method for synthesizing FDCA involves contacting DDG with an inorganic acid at a temperature in excess of 80° C. or 90° C. or 100° C. or HO ° C. or 120° C.
  • Another method for synthesizing FDCA involves contacting DDG with an inorganic acid under anhydrous reaction conditions.
  • the anhydrous conditions can be established by lyophilizing the DDG in any method of synthesizing FDCA disclosed herein so that the DDG contains less than 10% or less than 9% or less than 8% or less than 7% or less than 6% or less than 5% or less than 4% or less than 3% water or less than 2% water, by weight.
  • molar yields of FDCA can be obtained of greater than 10% or greater than 15% or greater than 20% or greater than 25% or greater than 30% or greater than 35% or greater than 40% or greater than 45% or greater than 50%.
  • Enzymes with natural activity for the dehydration of gluconate have been discovered (EC 4.2.1.39). Three enzymes from this family were cloned as shown in Table 1. Enzyme pSGI-365 was cloned and shown to be a dehydratase with broad substrate range having strong activity for the dehydration of gluconate (Kim, S. Lee, S. B. Biotechnol. Bioprocess Eng. 2008, 13, 436).
  • Proteins 359, 360, and 365 showed 2-5 ⁇ mole/min per mg of crude enzyme lysate activity for the synthesis of dehydration of gluconate (gel not shown).
  • Reaction buffer (93 mL) containing Kpi 10 mM pH 8.0 with 2 mM MgCl2 and 3.5 gr (0.016 mole) of sodium gluconate was mixed with 7 mL of the previous gluconate dehydratase solution. The reaction was incubated at 45° C. for 16 h before one aliquot was analyzed by HPLC-MS ( FIG. 4 ). As shown in FIG. 4 one new major product with the molecular weight of DHG was produced. The product was also shown to have activity with DHG dehydratases.
  • pRANGERTM Lucigen, Middleton, Wis.
  • pRANGERTM is a broad host commercially available plasmid vector containing the pBBR1 replicon, Kanamycin resistance and an pBAD promoter for inducible expression of genes.
  • pBAD promoter for inducible expression of genes.
  • SEQ ID NOs: 30-32 and 33-35 show the amino acid and nucleotide sequences, respectively, of the gluconate dehydratases #0385, #0336, and E3HJU7.
  • Enzymes of the family (EC 1.1.1.127) can be used to perform this step. Two examples are 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase and DHG dehydrogenases. Five enzymes from this family were cloned as shown in Table 2 below. pRANGERTM vector was used in every case.
  • the product prepared from the dehydration of gluconate in Step 2 was used as substrate for assaying the lysates of Table 2. As shown in the following Table 3, enzymes were identified showing activity for the oxidation of DHG in assays measuring NADH formation (absorbance increase at 340 nm).
  • Step 16 Further verification of the formation of 2,5-DDH by these enzymes was shown in Step 16 where the reduction of 2,5-DDH (made from the dehydration of 5KGA) with pSGI-395 at acidic pH was shown.
  • Steps 7 and 7B Conversion of Guluronic Acid to D-Glucaric Acid (7) and Conversion of L-Iduronic Acid to Marie Acid (7B)
  • Uronate dehydrogenases (EC 1.1.1.203) are enzymes that oxidize glucuronic and galacturonic acid.
  • Three enzymes with sequence similarity to the known uronate dehydrogenase (Expasy: ⁇ 7CRQ0; Prather, K. J, et al., J. Bacteriol. 2009, 191, 1565) were cloned from bacterial strains as shown in Tables 4 & 5.
  • Each protein was expressed with a His tag from pET28 and was purified prior to their screening. Protein gels of the crude lysates and purified enzymes are shown in the gel of FIG. 1 . After purification all enzymes were tested for activity against glucuronate, as well as against guluronate and iduronate. Kinetic measurements at different substrate concentrations were performed and the calculated activities and Km values for each enzyme are shown in Table 6. AU enzymes showed good activity for glucuronate, and also for L-iduronate and guluronate.
  • Each plasmid shown in Table 4 was transformed in BL21DE3 E. coli cells. Clarified lysates were mixed with equal volume of (25 mL) of equilibration buffer and purified on an Ni NTA column. Activity of each purified enzyme was measured in by mixing 0.050 mL of various dilutions of each purified enzyme with 0.95 mL of reaction buffer (100 mM TrisHCl, pH 8.0, 50 mM NaCl, 0.75 mM NAD+). The reaction progress was measured by monitoring of the formation of NADH at 340 nm.
  • reaction buffer 100 mM TrisHCl, pH 8.0, 50 mM NaCl, 0.75 mM NAD+
  • 6 a and 6 b provide Lineweaver-Burk plots for the oxidation of glucuronate and iduronate, with all three enzymes shown in FIG. 6 . Clear positive slopes were obtained with all enzymes giving the activities shown in the table above. Protein sequences of the uronate dehydrogenases are shown as SEQ ID NOs: 1-3 and the genes as SEQ ID NO: 4-6.
  • Step-15 Conversion of 5-ketogluconate (5-KGA) to L-Iduronic Acid (15) or Guluronic Acid (15A)
  • This example illustrates the identification of an enzyme capable of isomerizing 5-KGA to iduronic acid (Step 15) or guluronic acid (Step 15A). Thirteen enzymes from three different isomerase families were cloned as shown in Table 7, while their % sequence identity is shown in Table 8.
  • Activity for the isomerization of 5KGA and iduronate using enzymes from Table 7 was measured using an enzymatic method that detected the formation of products by their activity against two different enzymes.
  • isomerization of 5KGA was detected by measuring the activity of the product iduronate using uronate dehydrogenase (pSGI-476).
  • Isomerization of iduronate was detected by measuring the activity 5KGA reductase (pSGI-383, EC 1.1.1.69) of the product 5KGA. Presence of the products was also detected by GC-MS.
  • the purified isomerases were used in reactions using lysate and buffer containing 5KGA or Iduronate.
  • Product formation was demonstrating using both HPLC and the previously described enzymatic methods. Results for 17 h of incubation using both HPLC and enzyme assays are shown in FIG. 7 a . All enzymes showed good activity for the isomerization of both 5KGA and iduronate.
  • Yields for iduronate isomerization by pSGI433, pSGI 434, pSGI 435, and p SGI 436 were 56%, 48% 42%, (436 not measured), respectively when measured enzymatically and 78.8%, 78.5%, 733% and 76.6%, respectively when measured by HPLC assay. Yields after 16 h for 5KGA isomerization by the same enzymes were 18%, 17%, and 19% respectively (436 not measured) when measured by enzymatic assay, and 16.6%, 17.8%, 16.3%, and 16.9%, respectively, when measured by HPLC assay.
  • Enzymes from the EC 5.3.1.12 family were also purified by gel electrophoresis, isolated, and used to prepare reactions by mixing with buffer (50 mM HEPES, 1 mM ZnCl2, pH 8.0) that contained 5 mM of 5KGA or Iduronate. The reactions were incubated at 30° C. and analyzed for product formation using both HPLC and enzymatic methods. Results are shown in FIG. 7 b.
  • Enzymes pSGI-478 and pSGI-479 (5-dehydro-4-deoxy-D-glucuronate isomerases) showed isomerization activity for both 5KGA and iduronate. This activity was also confirmed with the enzymatic assays as above. Yields for isomerization of iduronate by pSGI-478 and -479 were 50% and 37%, respectively, when measured enzymatically, and 20% and 18% when measured by HPLC. Yields for 5KGA isomerization were 23% and 26%, respectively, when measured enzymatically, and 24% and 16%, respectively when measured by HPLC. Results are shown in FIG. 7 a.
  • Enzymes in this family were purified by gel electrophoresis. Product formation was measured using enzymatic assays as described above and the results are shown in FIG. 8 . All enzymes cloned in this family were shown to have activity for the isomerization of 5KGA and iduronate.
  • plasmids were transformed in BL21DE3 and proteins purified on a Ni NTA column.
  • Step 16 5-keto-gluconate (5KGA) to (4S)-4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH)
  • Example 1 The three gluconate dehydratases described in Step 2 (Example 1) were expressed as described in Example 1, along with a purified glucarate dehydratase from Step 8. Enzymatic reactions for activity were performed and HPLC-MS analysis showed the formation of 2,5-DDH ( FIG. 9 ), which was also confirmed by the fact that formation of the new product was accompanied by the reduction of 5-KGA only in the samples containing gluconate dehydratases, as well as by enzymatic assays with DHG dehydratase (pSGI-395). Good slopes at 340 nm indicating large enzyme activity were obtained when NADH, pSGI-395 lysate and aliquots of the previous reactions were mixed (data not shown). This result in combination with the HPLC analysis prove that the gluconate dehydratases examined dehydrate 5KGA to 2,5-DDH.
  • 1,5-gluconolactone oxidation is a side activity of enzymes from the alditol oxidases (EC 1.1.3.41) family. These enzymes oxidize various alditols such as sorbitol, xylitol, glycerol and others. Enzymes were identified having activity for the oxidation of 1,5-gluconolacone, as shown in Table 6 below.
  • Reactions were prepared using lysates of all the purified enzymes shown on Table 6. Reactions were prepared in 50 mM K-phosphate buffer, pH 7.0 with 0.5 mg/mL catalase and incubated at 30° C. A new product was observed by HPLC-MS analysis showing the same retention time as guluronate after comparison with authentic standards ( FIG. 10 ). This was confirmed by GC-MS, where the product also had the same MS fingerprint as guluronate. It is therefore clear that all the alditol oxidases described in the Table oxidize the 6-OH of 1,5-gluconolactone to produce the guluronic acid lactone. All alditol oxidases were cloned in pET28a with a HisTag and were expressed in BL21DE3 and purified on a Ni NTA column.
  • reaction solutions were combined and then diluted by pouring into ice (to neutralize the heat). Approximately equivalent volume of THF was added, and the solution transferred to a separation funnel. Sodium chloride salt was added until separation was achieved. The solution was agitated between additions for best possible dissolution. The aqueous layer was removed, and the THF layer washed 3 ⁇ more with sat. NaCL solution. Sodium sulfate was added and the solution left sitting overnight. Two layers formed again overnight. The aqueous layer was discarded and then silica gel was added to the solution. It was then concentrated down to solids via rotovap. The solids were loaded into a silica flash column and then separated via chromatographically. The fraction was concentrated and dried. The isolated yield was 1739 mg. Corrected yield: 24.9%. 1 H and 13 C NMR and HPLC-MS analysis confirmed the product
  • the invention provides a method for synthesizing a derivative of DDG.
  • the method involves contacting DDG with an alcohol, an inorganic acid, and optionally a co-solvent to produce a derivative of DDG.
  • the derivative of DDG can be purified.
  • the reaction can have a yield of the derivative of DDG of at least 10% molar yield or at least 15% molar yield or at least 20% molar yield or at least 25% or at least 30% or at least 35% molar yield or at least 40% molar yield.
  • the inorganic acid can be sulfuric acid and the alcohol can be methanol, ethanol, propanol, butanol, isobutanol, or any C1-C20 alcohol.
  • the co-solvent can be any of THF, acetone, acetonitrile, an ether, butyl acetate, an dioxane, chloroform, methylene chloride, 1,2-dichloroethane, a hexane, toluene, and a xylene.
  • the DDG derivative When the alcohol is ethanol the DDG derivative will be DDG mono-ethyl ester and/or DDG diethyl ester.
  • the alcohol is butanol the DDG derivative will be DDG mono-butyl ester and/or DDG dibutyl ester.
  • DDG mono-potassium salt was used for derivatization according to the following protocol.
  • a IL Morton type indented reaction vessel equipped with a mechanical stirrer and heating mantle was charged with 60:40 DDG:KCl (31.2 mmol), BuOH, and heptane.
  • sulfuric acid was added to water, and allowed to cool after dissolution. The solution was then added to the flask. The solution was kept at 30° C.
  • DDG esters such as mixtures of BuOH (5%-95% v/v) with co-solvents such as THF, acetone, acetonitrile, ethers (dibutyl, ditheyl etc), esters such as Butyl-acetate, 1,6-dioxane, chloroform, methylene chloride, 1,2-dichloroethane, hexanes, toluene, and xylenes may be used as cosolvents.
  • co-solvents such as THF, acetone, acetonitrile, ethers (dibutyl, ditheyl etc)
  • esters such as Butyl-acetate, 1,6-dioxane, chloroform, methylene chloride, 1,2-dichloroethane, hexanes, toluene, and xylenes may be used as cosolvents.
  • Reaction catalysts such as acids (sulfuric, hydrochloric, polyphosphoric or immobilized acids such as DOWEX) or bases (pyridine, ethyl-amine, diethyl-amine, boron trifluoride) or other catalysts commonly used for the esterification of carboxylic acids.
  • This example illustrates the enzymatic conversion of 5KGA to DDG using purified enzymes according to Scheme 6 (a sub-Scheme of 2B), and also illustrates the DDG produced being dehydrated to FDCA using chemical steps.
  • the Scheme involves the steps of isomerization of 5KGA (Step 15) and the subsequent oxidation to idaric acid (Step 7B). DDG was also dehydrated under differing chemical conditions to FDCA. The last step (Step-8A) was performed using glucarate dehydratase from E. coli.
  • Scheme 6 is illustrated in FIG. 11 .
  • the scheme was performed using a cell free enzymatic synthesis of DDG from 5-KGA.
  • the Scheme involves the performance of steps 15, 7B and 8A.
  • Two additional proteins were used to complete the reaction path, the first being NADH-oxidase (Step A) that is recycling the NAD+ cofactor in the presence of oxygen, and catalase (Step B) that decomposes the peroxide produced from the action of NADH oxidase.
  • the enzymes are shown in the following Table 7. All enzymes contained a HisTag and were purified using an Ni-NTA column. Yields for this synthesis of DDG were calculated to be at least 88-97%.
  • each reaction contained 50 mM TrisHCl (pH 8.0), 50 mM NaCl, 1 mM ZnCl 2 and 2 mM MgCl 2 , 1 mM MnCl 2 and 1 mM NAD + . Reactions were analyzed by HPLC after 16 h of incubation and FIG. 12 presents the chromatograms.
  • reaction mixtures of both samples were combined and lyophilized into a white powder, which was split into two samples and each dissolved in AcOH with 0.25M H 2 SO 4 or in 4.5 mL BuOH with 0.25M H 2 SO 4 . Both reactions were heated in sealed vials for 2-4 h at 120° C. Reaction products are shown in FIG. 13 .
  • Samples 1 and 2 represent authentic standard and the 3 h time point from the reaction in AcOH/H 2 SO 4 , respectively. Spiking of sample 2 with sample 1 gave a single peak further verifying the FDCA product.
  • Samples 1 and 3 ( FIG. 13 ) represent authentic standard and the 4 h time point from the reaction in BuOH/H 2 SO 4 , respectively. The formation of FDCA from the enzymatic reactions further confirms the presence of DDG in these samples.
  • This example shows the enzymatic conversion of glucose and gluconate to DDG.
  • the reaction was conducted with purified enzymes, and crude lysates as a catalyst. Enzymes and substrates were combined in a bio-reactor as shown in the Table below:
  • the following example describes the creation of recombinant nucleic acid constructs that contained coding sequence of a D-glucarate dehydratase activity (GDH, EC 4.2.1.40) for heterologous expression in E. coli cells.
  • GDH D-glucarate dehydratase activity
  • Each of the PCR-amplified genes was subsequently cloned into the bacterial transformation vector pET24a(+), in which the expression of each of the GDH genes was placed under control of a T7 promoter.
  • the nucleotide sequences of each of the PCR-amplified inserts were also verified by sequencing confirmation.
  • Each of the expression vectors constructed as described in Example 9 was introduced into NovaBlue(DE3) E. coli by heat shock-mediated transformation. Putative transformants were selected on LB agar supplemented with Kanamycin (50 ⁇ g/ml). Appropriate PCR primers were used in colony-PCR assays to confirm positive clones that contained each of the expression vectors.
  • a bacterial colony was picked from transformation plates and allowed to grow at 30° C. in liquid LB media supplemented with Kanamycin (50 ⁇ g/ml) for two days. The culture was then transferred into vials containing 15% glycerol and stored at ⁇ 80° C. as a frozen pure culture.
  • This Example describes how in intro synthesis of DDG intermediate was achieved using recombinant GDH enzymes produced in E. coli cells.
  • Recombinant bacterial strains constructed as described previously in Example 2 were grown individually in 3 mL of liquid LB media supplemented with Kanamycin (50 ⁇ g/ml) at 30° C. on a rotating shaker with rotation speed pre-set at 250 rpm for 1 day. This preculture was used to inoculate 100 mL of TB media containing Kanamycin (50 ug/ml), followed by incubation at 30° C. on a rotating shaker pre-set at 250 rpm for 2-3 hour until early log phase (OD 600 ⁇ 0.5-0.6) before isopropyl D-1 thiogalactopyranoside (IPTG; 0.25 mM final concentration) was added to induce protein expression.
  • Kanamycin 50 ⁇ g/ml
  • IPTG isopropyl D-1 thiogalactopyranoside
  • HPLC-MS results indicated a new peak as the only major product with a molecular weight corresponding to predicted product DDG, and trace amounts of the mono-potassium glucarate substrate. No other byproducts were detected by HPLC-MS analysis, indicating that the conversion reaction catalyzed by each of the recombinant enzymes was very efficient and highly specific.
  • DDG produced via enzymatic dehydration was purified by using either of the two following techniques.
  • the enzymatic dehydration reactions were acidified to pH ⁇ 2.0 with 6M HCl, filtered to eliminate precipitated proteins, and subsequently lyophilized.
  • Methanol Methanol
  • Substantially pure DDG acid was obtained following filtration of the suspensions and evaporation of MeOH.
  • FDCA i.e. the free acid form
  • H2SO4 a chemical conversion of DDG to FDCA in the presence of H2SO4.
  • the reaction was performed as follows. Approximately 20 mg of DDG acid (crude lyophilized powder with salts previously purified as described in Example 3) and 0.25 M of H2SO4 were added into an air tight sealed tube containing 1 mL of water and 1 mL of DMSO. The DDG was found completely dissolved in this solution. The reaction was stirred at 105° C. for 18 hours.
  • immobilized acids offer many advantages for performing dehydrations since they can typically operate in several types of solvent (aqueous, organic or mixed, etc.). In addition, they can be easily recycled and be re-used. Following some examples of the synthesis of esters of FDCA using immobilized AMBERLYST®15 (Rohm and Haas, Philadelphia, Pa.) and DOWEX®50 WX8 (Dow Chemical Co, Midland, Mich.).
  • FCA furfural-5-carboxylic acid
  • FCH furfural-5-carboxylic acid
  • transaminated using chemical reductive amination or transaminase
  • the inlet of the GC was used as a high temperature reactor to catalyze the dehydration of di-butyl DDG to di-butyl FDCA.
  • the resulting products were chromatographically separated detected by mass spectrometry.
  • a solution of di-butyl DDG (10 mM) and sulfuric acid (100 mM) in butanol was placed in a GC vial. The vial was injected into a GC and FDCA Dibutyl ester was observed. The reaction occurred in the 300° C. inlet (residence time 4 seconds). The average yield of 6 injections was 54%.

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